I doubt this will be of much use, but... Who knows if I'll be able to access links in a PDF, to get to sources one day?? Fun ideas!!! I know I read the crappy version of Harold and Kumar, last time.  I'm going to hell, for being turned into child porn anyway, and having a shitty life, most wouldn't take. Love you mom, sorry the drugs caused you to kill yourself and murder my brother. Sorry for all the bad advice others gave you!  Save the planet mak6a better world if anyone can.... Files above... I can't remember if I was able to access the website or just the text on it.... But ehhh, who cares.  Biochemistry, good luck! 

 

Available online at www.sciencedirect.com
ScienceDirect

journal homepage: www.elsevier.com/locate/he
Genetic engineering contribution to developing cyanobacteria-based hydrogen energy to reduce carbon emissions and establish a hydrogen economyGulzhanay K. Kamshybayeva a,b, Bekzhan D. Kossalbayev a,b,
Asemgul K. Sadvakasova a, Ardak B. Kakimova a, Meruyert O. Bauenova a, Bolatkhan K. Zayadan a, Chi-Wei Lan c, Saleh Alwasel d, Tatsuya Tomo e,
Jo-Shu Chang c,f,g,h,*, Suleyman I. Allakhverdiev i,j,k,l,**
a Faculty of Biology and Biotechnology, Al-Farabi Kazakh National University, Al-Farabi 71, Almaty 050038,
Kazakhstan
b Department of Chemical and Biochemical Engineering, Institute of Geology and Oil-Gas Business Institute Named after K. Turyssov, Satbayev University, Almaty 050043, Kazakhstan
c Department of Chemical Engineering and Materials Science, Yuan Ze University, Chung-Li 32003, Taiwan
d College of Science, King Saud University, Riyadh 12372, Saudi Arabia
e Department of Physics, Graduate School of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku,
Tokyo 162-8601, Japan
f Department of Chemical and Materials Engineering, Tunghai University, Taichung 407, Taiwan
g Research Center for Smart Sustainable Circular Economy, Tunghai University, Taichung 407, Taiwan
h Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan
i K.A. Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, Moscow 127276, Russia
j Institute of Basic Biological Problems, FRC PSCBR RAS, Pushchino 142290, Russia
k Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
l Faculty of Engineering and Natural Sciences, Bahcesehir University, Istanbul, 34353, Turkey

h i g h l i g h t s

Genetic engineering findings of cyanobacterial nitrogenase enzyme are described.
Recent genetic engineering methods to obtain hydrogenase mutants are discussed.
Engineering photosynthetic H2 production is shown as an efficient method.
Development of a non-waste H2 production technology is explained.

 

a r t i c l e i n f o

Article history:
Received 1 November 2022 Received in revised form
a b s t r a c t

Growing concerns over greenhouse gas emissions and energy insecurity caused by the depletion of conventional fuels have led to a search for sustainable fuel alternatives. As an alternative energy carrier, hydrogen (H2) is particularly attractive as only water is released

 

* Corresponding author.
** Corresponding author.
E-mail addresses: changjs@mail.ncku.edu.tw, changjs@thu.edu.tw (J.-S. Chang), Suleyman.allakhverdiev@gmail.com (S.I. Allakhverdiev).
https://doi.org/10.1016/j.ijhydene.2022.12.342
0360-3199/© 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

 

26 December 2022
Accepted 29 December 2022 Available online xxx

Keywords: Biohydrogen Photosystem Cyanobacteria Nitrogenase Hydrogenase
during combustion. The process of H2 production from genetically engineered photo- trophic microorganisms through biophotolysis leads the way to solve energy shortages. Genetically engineered cyanobacteria species are potential candidates due to their superior properties for reducing greenhouse gases and using solar energy as an energy source. The review discusses the mechanisms and enzymes involved in H2 production by cyanobac- teria and applications of genetic engineering. A critical analysis of the fundamental issues attributed to the technical advancement of photobiological cyanobacteria-based H2 pro- duction is provided, as well as the perspectives for future research to reduce carbon dioxide emissions through the creation of waste-free technology.
© 2022 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

 

 

 

Introduction

The major environmental challenge of the new millennium is global warming and air pollution caused by fossil fuel burning. This problem has necessitated the search for new renewable energy sources that are non-polluting and could replace non- renewable energy sources when they become scarce and more expensive [1]. Therefore, scientists focus on producing bio- fuels from secure, inexhaustible feedstocks, such as hydrogen (H2), by photocatalytic decomposition of water (H2O). Among the various gaseous and liquid biofuels, H2 is one of the most valuable and cleanest fuels, and it may serve as an environ- mentally safe and forthcoming renewable energy carrier [2]. As of today, H2 is primarily produced by thermochemical processes [3]. However, biological H2 production is of great scientific and practical relevance since it avoids the use of fossil fuels, whose reserves are diminishing each year, and the environmental pollution resulting from using conventional energy sources [4e6]. In conjunction with the issue related to energy production, the intermittency of renewable energy sources is another point to be considered, from which the necessity to accumulate and store part of the H2 energy pro- duced emerges. Although there are several excellences in the field of photovoltaic PV and H2 storage around the world, both at the academic and industrial levels, only a portion of the scientific community has prioritized the appropriate and innovative integration of energy generation and storage de- vices [7e10].
Currently, in numerous research studies, cyanobacteria are being investigated as a solution to such environmental problems [11,12]. Moreover, only cyanobacteria are capable of performing oxygenic photosynthesis among prokaryotes. Solar energy, H2O, carbon dioxide (CO2), and mineral nutrients are used to create a considerable amount of oxygen (O2) and organic assimilation for an aerobic food chain [9]. In addition, cyanobacteria are considered prospective “low-cost”” cell factories to capture and store carbon, as well as to produce
separated O2 and H2 evolution processes and producing H2 in an O2-contained environment can carry out light-dependent H2 production. This process is appealing because both solar energy and the substrate (water) are inexhaustible and renewable, and the by-product (O2) is non-toxic [17]. The ge- netic ability to synthesize the essential enzymes, as well as the internal and external metabolic and environmental con- ditions that supply the necessary energy, control the rate of cyanobacteria-based H2 production.
A growing variety of molecular tools has recently unleashed the potential of cyanobacteria [18]. These ad- vancements have coincided with an increase in the ability to modify endogenous genetic sequences and transfer exoge- nous DNA into a wide range of cyanobacterial strains [19,20]. Furthermore, breakthroughs in sequencing technology have enabled the genomes of over 200 cyanobacteria to be sequenced [21], considerably simplifying the use of system- level approaches such as transcriptomics and proteomics. With the help of new sequencing tools, modification of the genes of the nitrogenase enzyme, obtaining hydrogenase mutants, increasing electron transport by changing the photosynthesis complex, and obtaining mutants resistant to external environmental factors can be the most effective methods of biological H2 production. Three main aspects must be considered simultaneously to produce H2 from modified cyanobacterial cells: technological limitations, metabolic characteristics, and genetic engineering [22e24] (Fig. 1).
Although many cyanobacterial species worldwide have been studied in the laboratory for H2 production, high H2 amounts have not yet been obtained under industrial condi- tions. In this context, this review analyzes the main ways of obtaining promising H2 production mutants that can be used for industrial purposes and evaluates their economic and so- cial effects. Next, an examination was made to reduce the amount of CO2 in production fields by creating a zero-waste technology for H2 energy production based on cyanobacte- rial biomass.

sustainable biofuel, due to the basic nutritional requirements,

physiological stability (as they can colonize many biotopes, transportation costs can be potentially reduced by enabling industrial production nearby), and strong genome-based properties of some model strains [13,14]. It should be noted that cyanobacteria are up-and-coming H2 producers consid- ering their morphological and metabolic characteristics
[15,16]. Indeed, heterocystic cyanobacteria having spatially
Cyanobacteria-based H2 production enzymes
Two groups of cyanobacterial enzymes are involved in H2 metabolism: nitrogenases (N2ase) and hydrogenases (H2ase) [25]. Nitrogenases are typically present in unicellular or fila- mentous cyanobacteria that fix nitrogen (N2) and reduce it to ammonium ion (NHþ), thereby producing H as a by-product

4 2

 

Fig. 1 e Pathways to increase H2 cleavage in cyanobacterial strains.

 

[26]. Furthermore, two types of cyanobacterial [NiFe]-N2ases, namely bidirectional and uptake N2ases, are found. The dif- ference between bidirectional N2ase and bidirectional H2ase (reabsorbs H2 produced by N2ase) is that the latter is found primarily in diazotrophic microorganisms [27].

heterocyclic filamentous cyanobacteria spatially engage in O2 evolving photosynthesis (in vegetative cells) and N2 fixation (in heterocysts with an O2 deficiency) [28,29];
heterocystic cyanobacteria accomplish N2 fixation and arecapable of H2 production, usually under O2 deficiency, rarely under O2-rich conditions (e.g., by managing the
daily circadian rhythm or intracellular sections) [30].
ends. The nifH gene encodes the small homo-dimeric (g2) protein. The significant component, which is a tetrameric (a2b2) protein, has a molecular weight of 240 kDa and binds to each ab dimer. Two types of molybdenum metal-dependent nitrogenase enzymes have been found in cyanobacteria. The first enzyme (nitrogenase, encoded by the nif1 gene cluster) is activated in heterocysts; the second enzyme (encoded by the nif2 gene cluster) functions only under anaerobic/anaerobic conditions in vegetative cells.
In general, all H2ase types follow the following basic for- mula (eq. (1)):
16ATP þ 16H2O þ N2 þ 10Hþ þ 8e- / ðN2aseÞ 16ADP

þ 16Pi þ 2NH4 þ H2
(1)

Nitrogenases
The primary function of N2ase is to convert atmospheric N2 into NHþ;H production is negligible, considering it a negative

4 2

Nitrogenases function differently, depending on the structure of the clusters they contain, and genes directly control the function of the clusters within them. The overall structure of N2ase consists of two parts [31]: while the di-N2ase protein occupies the first part, the second part consists of the di-N2ase reductase protein. The di-N2ase is an a2b2-heterotetramer
(Mr ¼ 220e240 kDa) that splits N2 atoms [4], whereas the di-
N2ase reductase is a homodimer (Mr ¼ 60e70 kDa). The pro- tein's primary function is transporting electrons from the
external environment (ferredoxin) to di-N2ase. Three distinct di-N2ase proteins have been identified, each with a unique metal cofactor. The first type contains molybdenum (Mo), the second is vanadium (V), and the third type is iron (Fe) [32].
In cyanobacteria, Mo nitrogenase consists of two proteins (Fe protein and MoFe protein), which are connected by their
and undesirable side reaction [33]. Nitrogenases exclusively catalyze H2 production on N2-free substrates [34]. In native, intact cyanobacterial cultures, H2 production by N2ases is functionally tied with H2-scavenging Hup-H2ase, leading to zero net H2 production. However, by changing certain envi- ronmental conditions, such as switching from an O2-rich to an O2-free phase, a considerable H2 yield might be achieved [35]. As shown in Fig. 2, cyanobacteria can be divided into three categories depending on the H2 enzymes they contain.

multicellular cyanobacteria (Synechocysts sp. PCC6308) with [NiFe)-hydrogenase (bidirectional hydrogenase) located only in vegetative cells;
types of filamentous cyanobacteria (Anabaena sp. PCC7120) with [NiFe)-hydrogenase (bidirectional and uptake hydrogenases) in vegetative cells and

 

Fig. 2 e Mechanisms of H2 production in different cyanobacterial species. Modified from Refs. [34,35]. Abbreviations: PSI e photosystem I; PSII e photosystem II; NDH-1 e NAD(P)H dehydrogenase complex; PQ pool e plastoquinone pool; Cyt b6f e cytochrome b6f complex; PC e plastocyanin; Fdx e ferredoxin; FNR e ferredoxin-NADP(þ) reductase; NADPH e nicotinamide adenine dinucleotide phosphate; CO2 e carbon dioxide; ATP e adenosine triphosphate; ATPase e adenosine triphosphatase; Flv e flavodiiron proteins; Hþ e protons; OPP pathway e oxidative pentose phosphate pathway; N2ase e nitrogenase; H2ase e hydrogenase.

 

nitrogenase and bidirectional [NiFe)-hydrogenase in heterocyst cells;
multicellular cyanobacterial species with active bidi- rectional [NiFe)-hydrogenase and weak nitrogenase (Cyanothece sp. ATCC 51142).

Hydrogenase

Hydrogenase is the second most abundant enzyme involved in H2 production in cyanobacteria. Many cyanobacteria contain various types of H2ase enzymes. Uptake H2ases (encoded hupSL) can oxidize H2, while the bidirectional H2ase (encoded hoxFUYH) enzyme can absorb or produce H2 [36]. Uptake H2ase is mainly observed in the thylakoids of fila- mentous bacterial heterocysts. Oxy-hydrogenation, or the Knallgas reaction, implies a transfer of electrons from H2 via the respiratory chain to perform O2 reactions [37]. This enzyme is composed of two subunits. The hupL-encoded protein functions as an uptake H2ase, while the hupS-encoded component carries out the reduction process (Fig. 2). As the resulting H2 is usually re-oxidized by H2ase (according to the
Knallgas reaction), strains containing uptake H2ase do not produce pure H2 under environmental conditions [38]. Thus, if H2 production needs to be scaled up to an industrial scale, a considerable amount of net H2 production is required between the uptake and H2-producing H2ases. The uptake H2ase cata- lyzes the reaction, which follows the formula (eq. (2)) [39]:
H2 / ðH2aseÞ 2Hþ þ 2e- (2)
The biological function of bidirectional and uptake H2ases is not considerably clear, and maintaining ionic concentration in cells could be a possible purpose. Uptake H2ase is bound to the cytoplasmic membrane and serves as an electron acceptor for NADH and H2 [40]. Reversible H2ase is a multimeric enzyme consisting of four or five (depending on the species) different subunits. It is encoded by the hoxYH gene, which controls [NiFe]-H2ase. The activity of auxiliary proteins known as hyp is required to develop bidirectional H2ase [41]. Contrary to the uptake H2ases, the bidirectional ones are directly involved in H2 production [42].
[FeFe]-hydrogenases are another type of hydrogenase found in microorganisms. This type of hydrogenase has two different cofactors: iron and sulfur. In addition, this enzyme is

 

attached to the protein skeleton by three cysteine residues. The iron cluster is connected to the di-iron part by a fourth cysteine residue. Hydrogenase in this cluster is not found in cyanobacteria. However, as a group of foreign genes, it can activate in cyanobacterial cells.
An anoxic environment is created in cyanobacterial cells by separating the processes of O2-evolving photosynthesis and O2-sensitive enzymes spatially or temporally, in combi- nation with efficient cellular respiration [43]. The phosphor- ylation process that generates the necessary ATP energy requires the bidirectional H2ase found in about half of all cyanobacteria. This evolutionary process demonstrates that bidirectional (hox) H2ase has functions other than cell-specific H2 separation [44]. Many cyanobacterial species, however, can live freely in nature without bidirectional (hox) H2ase and are competitive with other organisms. The biological functions of bidirectional (hox) H2ase in cyanobacteria include cellular redox processes, such as the release of energy stored as H2 to the external environment under stress conditions, ensuring cell safety [35,45].
Several known cyanobacterial H2 production reactions can
be utilized in large-scale production [46]. While growing in an alternating dark light mode (i.e., photosynthesis and respira- tion occurring under circadian regulation in response to light similar to a sleep-wake cycle), some unicellular and filamen- tous cyanobacteria (e.g., Spirulina sp. [47], Synechocystis sp. RF-1 [48], Cyanothece sp. Atcc51142 [49], Desertifilum sp [31]. and Oscillatoria sp [31]) contain an active N2ase. The circadian rhythm regulates N2 fixation, photosynthesis, and respiration and prevents O2 from inhibiting N2ase. N2 fixation via N2ase catalysis is primarily observed in a wide range of heterocyst- forming cyanobacteria and azotobacter [50]. However, the strain Cyanothece devoid of heterocysts can fix N2 using light by N2ase. The circadian rhythm is most often observed in a dark environment. The cell is considered to revert to a reduced state when photosynthesis resumes in the light. When O2 release occurs, however, this activity is rapidly suppressed [51].

Genetic engineering of cyanobacterial enzymes for efficient H2 production

The use of modern methods to increase or eliminate the desired characteristics of wild species is called genetic engi- neering [52]. Genetic engineering methods are mainly imple- mented via the transfer of genes [53]. Transcription and translation processes are implemented using advanced syn- thetic biology techniques [54]. Despite scientific advances, only about 2000 species of cyanobacteria have been fully sequenced, including Cyanothece sp. ATCC 51142, Synechocystis sp. PCC6803, Anabaena sp. PCC 7120, and Synechococcus sp. PCC 7942 [55e57]. As whole gene groups have not yet been iden- tified and only partial sequencing has been conducted, the remaining cyanobacterial species are unsuitable for genetic studies and modifications (Fig. 2). Natural, artificial, causative, and inducible promoters are required to regulate the tran- scription of heterologous genes [58]. Non-natural (orthogonal) promoters do not affect cellular regulation or metabolism [59]. The capacity of cyanobacteria to produce H2 directly from solar energy and H2O is of great interest to the scientific
community [60]. However, photobiological H2 production by natural cyanobacteria faces several challenges, including inefficient use of solar energy, enzyme O2 sensitivity, and inefficient metabolism [61]. In general, the highest production of a particular molecule or product from biological systems is typically uncommon, and competition and survival exert evolutionary constraints on natural cyanobacteria [62]. Natu- ral cyanobacterial cells that produce and release H2 lose en- ergy and electrons, negatively influencing their competitive environment in situ [63].
Exploring the genetic control of H2 production and using genetic approaches to obtain efficient, productive strains is one way to optimize the process of bio-H2 production [64]. Therefore, creating genetic mutants to obtain photobiological H2 is economically viable and the only way to achieve a higher yield in the future [65]. Genetic approaches to increasing H2 yield by phototrophic microorganisms, including cyanobac- teria, include gene knockout, insertion, and overexpression [66]. Thus, H2 evolution in some cyanobacterial species is significantly higher after genetic modification than in wild species. N2ase and H2ase enzyme engineering are primary genetic engineering approaches to increase H2 yield. Studies on H2ase enzymes are primarily concerned with eradicating or inactivating the uptake H2ase and generating NH4-insensitive mutants. In contrast, studies on N2ase enzymes are concerned with increasing the number of heterocysts in filamentous cyanobacteria (Table 1) [67].

Genetic engineering of the nitrogenase

4
Nitrogenase converts molecular nitrogen into NHþ while releasing H2. For the following reasons, the improvement ofan N2ase-based H2 production system may be of particular interest.

N2ase biochemistry and proteins crystal structures are well-studied and available [78];
N2ase in heterocysts is naturally protected from O2 released during photosynthesis in PSII (photosystem II) [79];
some filamentous cyanobacteria of the order Nostocales (Nostoc, Anabaena, etc.) can fix carbon via photosynthesis and simultaneously release H2 using N2ase [80];
at high partial pressure, the enzyme can produce H2 [81].

4 2 2
H2ase-mediated H2 production lacks the above features. However, H2ase engineering is also promising to boost cyanobacteria-based H2 yield [82]. N2ase converts atmo- spheric N2 to NHþ and protons to H [83]. The N ase system is the principal source of H2 during photosynthesis, and N2 limitation occurs in many photosynthetic bacteria. Moreover, bio-H2 production efficiency is heavily reliant on N2ase, as well as the availability of ATP and electrons in the cell [62,84]. Skizim et al. [85] evaluated the ratio of H2 produced by N2asecompared to H2ase in Cyanothece Miami BG 043511 (light-vs. dark-induced). This species is capable of actively releasing hydrogen during illumination. Therefore, the transition be- tween light and dark and the intensity of light have a direct effect on the amount of hydrogen produced. Moreover, the generation of the N2ase mutant library by random

Table 1 e Hydrogen production in engineered native enzymes of cyanobacterial strains using different strategies.
Cyanobacterial strains
Engineered genes
H2 production rate
H2 production assay condition
References
Synechocystis PCC6803
HoxW
4.5 nmol H2 10-9 cells s-1
2500 lux; 31.25 mE m-2 s-1, on BG-11
[24]

 

 

medium enriched with 3.78 mM Na2CO3

Anabaena variabilis AVM13
hupSL
135 mmol H2 mg Chl a-1 h-1
Ar; 100 mEm-2 s-1; N2-fixing
[68]
Nostoc punctiforme NHM5
hupL
14 mmol H2 mg Chl a-1 h-1
Light and N2-fixing
[69]
Anabaena sp. PCC 7120
hupL/hoxH
53 mmol H2 mg Chl a-1 h-1
Ar; 10 Wme2; N2-fixing
[70]
Nostoc sp. PCC 7422
hupL
100 mmol H2 mg Chl a-1 h-1
Ar þ 5% CO2, 70 mEm-2 s-1; N2-fixing
[71]
Anabaena siamensis TISTR 8012
hupS
29.7 mmol H2 mg Chl a-1 h-1
Ar; 200 mEm-2 s-1; N2-fixing
[72]
Synechocystis M55
ndhB
200 nmol H2 mg Chl a-1 h-1
Anaerobic and N2 deprivation
[73]
Synechocystis sp. PCC 6803
ctaI/cyd
190 nmol H2 mg Chl a-1 h-1
Anaerobic and N2 deprivation
[74]
Synechococcus sp. PCC 7002
ldhA
14.1 mol H2 day-1 1017 cell-1
Dark anaerobic fermentation
[75]
Anabaena sp. PCC 712
hupW
3.3 mmol H2 mg Chl a-1 h-1
Dark anaerobic fermentation
[76]
Cyanothec 51,142
hupS
373 mmol H2 mg Chl a-1 h-1
Photoautotrophic growth under ambient
[77]

 

 

CO2 concentrations, argon incubation

Anabaena 29,413 (PK84 mutant)
hup-
167.6 mmol H2 mg Chl a-1 h-1
2% CO2, 24 argon incubation

Synechocystis 6803 (M55 mutant)
ndhB-
56 mmol H2 mg Chl a-1 h-1
Glucose, glucose oxidase, sulfur

 

 

 

deprivation, argon incubation

 

 

mutagenesis and gene shift could significantly contribute, although developing high-throughput screening methods is required. Furthermore, understanding the biochemical criteria for efficient H2 synthesis by [Mo]-N2ase is essential as a foundation for its re-engineering [85].
This is because mutants obtained by these methods are not NH4-sensitive, which increases H2 yield [94]. In the case of specific residual mutant strains that are not NH4-sensitive, the cyanobacterial mutant strains obtained in this manner may have advantages when using wastewater with very high NHþ

4
The N2ase enzyme requires an O2-free environment to function properly [86]. This process can occur in cyanobac- teria under two conditions: transiently in unicellular cells and permanently in filamentous cells, where O2 accumulates as a result of photosynthesis in vegetative cells. As a result, fila- mentous cyanobacteria can fix N2 in free space without re- striction and form an efficient respiratory system [87]. Filamentous cyanobacteria (Anabaena, Nostoc, etc.) are domi- nant in N2- and O2-free environments. Heterocysts contribute 5e10% of vegetative cells. This process occurs in response to N2 deficiency to maintain the vital activity of cells [88]. Moreover, H2ase is sensitive to O2 and N2. Studies show that many filamentous cyanobacterial heterocysts form in N2-free environments, although heterocysts also form in nitrogen- rich environments [89,90]. N2ase occurs in heterocysts, and some studies have found it in multicellular species of Oscil- latoria, Desertifilum, and Synechococcus [91].The following genetic engineering studies are conducted in conjunction with obtaining NH4-insensitive mutants. The de- gree of N2 fixation by filamentous cyanobacteria is based on the highly effective regulatory genes responsible for NH4 concen-
tration. A high NHþ concentration hinders the process of H
concentrations [95].
Another study on genetic engineering of the enzyme N2ase was conducted by transferring 35 genes of the nif cluster (nifH, nifD, nifK, etc.) from the strain Cyanothece sp. ATCC 51142 to Synechocystis sp. PCC 6803. The O2 resistance of H2ase in Syn- echocystis 6803 was improved by the introduction of N2ase reception genes, which showed that this was a functional way to enhance the activity of H2ase under microoxic conditions. As a result, the N2 fixation capacity of the Synechocystis sp. 6803 strain was 30% higher than in wild species, which is the highest activity found in a non-diazotrophic photosynthetic organism [96]. Furthermore, Li et al. [97] showed that the suf operon electron transport genes of Paenibacillus sp. WLY78 can increase H2ase activity. Therefore, about 28 genes were selected from WLY78 and expressed in recombinant E. coli 78e7. The derived mutant cells produced 50.1% more H2 than the wild-type cells.

Genetic engineering of heterocyst formation
Certain N2-fixing filamentous cyanobacteria develop hetero- cysts under N2 deficiency. Heterocysts have stronger cell walls than the neighboring vegetative cells, lack PSII, and show

4 2

4
cleavage due to the suppression of N2ase catalysis and the discontinuation of its activity. A reduction in nitrogenase ac- tivity could boost H2 production by lowering the sensitivity of N2ase to NHþ ions [92]. Point mutations in the nifA gene orknockout of the glnA (glutamine synthetase) gene make the
NifA protein NH4-insensitive and lead to average H2 production and N2 fixation even in an NH4-containing environment, which can yield such outstanding outcomes [92]. With 2- and 4-mM ammonium concentrations in the medium, the wild-type cells produced 1558 ± 12 mL L-1 of H2. Mutant cells produced 1678 ± 57 mL L-1 of H2, much higher than the wild type [93].
4
Furthermore, N2ase activity can be increased or normal- ized by modulating the genes that transport NHþ into the cell. enhanced respiratory activity, resulting in microoxic condi- tions that preserve O2-sensitive N2ases [45,98]. Fdx reduced by PSI yields electrons for N2 fixation. Clostridial [FeFe]-H2ases produced in heterocyst cells can scavenge photosynthetic electrons before they reach the N2ase and produce a higher H2
yield (0.22 mmol H2 dry-1 wt h-1) at a rate exceeding that of the
original strain [46]. However, [FeFe]-H2ase proved to be more O2-sensitive than N2ase and was even inactivated in hetero- cysts. The Flv3B gene (encoding a flavo-diiron protein) over- production boosted heterocystic H2 production, most likely via increased O2 consumption, and may promote the expres- sion of [FeFe]-H2ases as well. However, H2 production in het- erocysts is laborious and needs electrons and carbon

 

skeletons from functioning vegetative cells, which is less efficient than direct photolysis [98].
The process of active H2 evolution in N2-fixing cyanobac- teria is accompanied by an increase in heterocyst numbers in the filaments. The number of heterocysts can be artificially increased, which raises the concentration of N2ase [99]. This method can now be implemented by treatment with 7- azatriptophan or other genetic methods. Therefore, key genes involved in or controlling the formation of heterocysts need to be deciphered [100]. By over-expressing or introducing these genes, the number of heterocysts in the filaments can be regulated. It is currently known that more than 1000 genes directly or indirectly influence the formation of heterocysts. All genes mainly belong to the group of het genes, of which the hetR gene is the most actively studied. While the hetR gene is responsible for heterocyst formation, the gene products of patS and hetN [101] are mainly responsible for heterocyst elimination. So far, numerous researchers have investigated the turnover of heterocysts under various conditions. For example, Buikema et al. [102] increased heterocyst formation in Anabaena PCC 7120 by 30% by regulating the hetR gene, although this number decreased to 30% under N2 starvation.
During heterocyst formation, the HetR (serine protease)
and PatA (responsible regulator) genes impart signals that are crucial for the differentiation process [103]. However, there are no comprehensive studies on the production of bio-H2 by genetically modified free-living cells to increase heterocyst content. Although such elements can theoretically produce a higher yield of H2, they still need to be investigated. However, despite continued research efforts, N2ase-based H2 produc- tion requires increased energy in the form of ATP: for each mole of H2 produced, at least 4 mol of ATP are needed [104].
The patS and hetN genes are often used to increase the activity of N2ase. Moreover, they have been shown to influ- ence the structure of heterocysts indirectly. For example, black et al. [105] revealed that the hglK gene's main function was to form the outer, thick layer of the heterocysts. The thick layer stimulates the N2ase enzyme by blocking O2 passage from the external environment to the heterocysts. It was observed that, in cells deprived of the hglK gene, H2 yield decreased sharply.
As mentioned earlier, heterocyst formation in filamentous cyanobacteria in an O2-free environment affects the N2 cycle [106]. However, the process of heterocyst formation is not fully understood, although some studies suggest that heterocyst cells arise from older vegetative ones. Interestingly, free-living cyanobacterial strains show a heterocyst abundance of 5e10%, while modified strains gradually increase to 30e35% with a concomitant increase in N2 fixation. Moreover, the mutant strains increase the transport of cellular products by performing high levels of N2 fixation [107]. The H2 content of DnblA mutant filamentous cyanobacterium Anabaena sp. 33,047 grown in bioreactors is 2e10-fold higher (100 mm H2
mg-1 Chl a h-1) than that of wild species, and the number of
heterocysts increases. Genetically modified cyanobacteria can be ideal model systems for effective cell development that can be used in future photobioreactors. This is due to the avail- ability of various theories and genetic methods that control the differentiation of heterocysts.
Furthermore, enhanced H2 production requires the sepa- ration of metabolic H2 from microbial growth by increasing heterocyst abundance. Jeffries et al. [108] physiologically established constant H2 production in Anabaena cylindrica under N2-deficient conditions. In heterocyst cells, H2 was produced using stored carbohydrates and PSI. In addition, ferredoxin and RNR play a crucial role in cyanobacterial H2 yields [109]. In addition, some studies have shown that the Fe content of the culture medium positively influences hetero- cyst formation. The abundance of heterocysts is significant as they serve as a chassis for H2 production [106].
Only [NiFe]-H2ases that can be active during ethylene and H2 production by the N2ase enzymes are found in heterocyst cells. Introducing non-native [FeFe]-H2ase to cyanobacteria cells resulted in a higher H2 yield than its wild type. One of the most significant tasks in bio-H2 research is introducing H2- producing [FeFe]-H2ase into heterocysts of filamentous diaz- otrophic cyanobacteria to boost H2 production [46]. Despite the incorporation of clostridial [FeFe]-H2ase into Nostoc sp. PCC 7120, sufficient levels of H2 production have not yet been attained. When Fe-limited filaments are effectively converted into heterocysts, [FeFe]-H2ase requires Fe during Fe-limited culture. [FeFe]-H2ase requires the same amount of Fe as other H2ases: more than 10 Fe atoms for every functional H2ase protein complex [46]. Therefore, all heterologously expressed H2ases can influence the photosynthetic Fe requirement and remaining Fe-containing enzymes such as N2ase [110]. Longstanding Fe deficiency (~90 days) in wild-type Nostoc PCC 7120, on the other hand, revealed that the Fe ho- meostasis mechanism is tightly controlled and maintains photosynthesis and N2 fixation at a reduced but stable level after a few days of culture. Given H2ases fast turnover rate [111], the primary goal should be a well-regulated expression level that fits the metabolic capability of H2 production while minimizing the metabolic burden of H2ase on available intracellular Fe.
Moreover, a robust process of H2ase activation can be
implemented at the transcriptional level to synchronize expression with the onset of Fe-limiting effects. Since N2ase activity naturally decreases under Fe deficiency [112], the remaining Fe used for N2 fixation can be redistributed to [FeFe]-H2ase. On the other hand, a basic level of N2 fixation is required to stabilize the culture and maintain the proteome, i.e., the PSII repair mechanism. Therefore, Fe-limited cultures can be used to support heterocyst-based H2 production while the activity of N2ase remains negligible. Since Fe-limitation inhibits the growth of cyanobacterial cells and increases intracellular carbohydrate storage, this is a promising culture strategy for utilizing energy stored photosynthetically for H2 biosynthesis. Rather than enabling cyanobacteria to grow continuously in photobioreactors, as has been advocated in the last 30 years of biofuel research [113], solar energy should be converted directly into H2 or other fuels.
Genetic engineering of heterocysts can be performed to
increase their abundance, the thickness of the cell membrane, the amount of sugar coming from the vegetative cell, and the speed of arrival. In addition, the modification of genes contributing to the formation of heterocysts in a nitrogen or oxygen environment is also of interest.

 

Construction of the O2-resistant hydrogenase

Biocatalysts will play an increasing role in processes aimed at providing alternative and clean fuel sources [114]. However, producing enzyme cells in the presence of hydrogenases is currently difficult because O2 inhibits their activity. The enzyme sensitivity to O2 is the main impediment that must be addressed to develop effective photobiological H2 production. [NiFe]-H2ases are cyanobacterial hydrogenases that include both uptake Hup-H2ase (2Hþ / H2) and bidirectional Hox-
H2ase (2Hþ ⇔ H2). Common [NiFe]-H2ases are resistant to microaerobic conditions and are only transiently inhibited by
O2. Several microorganisms with O2-resistant [NiFe]-H2ases, such as Ralstonia eutropha, have O2-resistant H2ases that can oxidize H2 in the presence of atmospheric pO2 [115]. Although not all species can accommodate these plasmids, the research on transporting genes encoding natural proteins in this or- ganism to the necessary cyanobacteria appears very prom- ising [116,117]. The genes responsible for the growth of the bacterium R. eutropha in a microanaerobic medium were fully studied. Nevertheless, R. eutropha H2ase is considered a model system to inspire the development of effective O2-resistant hydrogenases [118].
Genetic engineering techniques to increase H2 synthesis by shifting electron flow towards H2 metabolism have recently received much interest. An engineering approach to removing competing electron routes, such as the respiratory ETC, the nitrate absorption system, and C fixation via the Calvin- Benson cycle, might be a potential technique to improve H2 production. Thus, decreasing nitrate uptake reduces electron flow for H2 metabolism, while increasing its yield. Synechocystis PCC 6803 DHar and DHir mutants (deletions of the DHar and DHir genes) with impaired nitrate uptake produced a higher H2 yield. Furthermore, inactivating hiloxidase in Synechococcus PCC 7002 increased H2 production in vivo [97]. According to
McNeely et al. [75], increasing the NADPH/NADP þ ratio also
increases H2 production by NADPH-dependent, bidirectional [NiFe]-H2ase. H2 synthesis can thus be enhanced by lowering or removing competing metabolic processes.
Fritsch et al. [119] presented a novel crystalline biological active substance (membrane-bound H2ase from R. eutropha H16-MBH). This crystal enters the thylakoid membrane and retains O2 without allowing it to enter the domain of enzymes that cleave H2 molecules. Since there is no O2-resistant component in the structures of [NiFe]-H2ases, the crystal structure of O2-resistant H2ase is a mechanism used by some of these enzymes to protect them from O2, and this control may pave the way for H2ases in many biotechnological de- velopments. Goris et al. [120] also engineered a membrane of
R. eutropha H16 that allows the O2-resistant [NiFe]-H2ase to produce H2 in an O2-rich environment. The authors demon- strated that O2 resistance is critically related to changes in the internal ETC. Six instead of four conservative coordination cysteines surrounded the [FeeS] cluster near the active site. O2 tolerance is not only based on the limited O2 access to the active site but also on reducing O2 species guided by the electronic relay's unique structure.
Genetic engineering of O2-resistant hydrogenase enzymes
is carried out by modification of their responsible genes. In this context, studies aimed at reducing the amount of O2
released by PSII in an anoxic environment will undoubtedly bear fruit. The hydrogenase enzyme can be made O2-resistant by replacing the cluster.

Engineering of [NiFe]-hydrogenase to enhance hydrogen production
The indicators of H2 production by cyanobacteria species ob- tained from many natural fields have been studied, and opti- mization work has been carried out in laboratory conditions. However, despite the efforts made for half a century, indus- trially useable H2 has not yet been isolated from cyanobac- teria. In this context, the importance of obtaining mutant strains through genetic engineering before implementing metabolic and technical strategies is increasing. [NiFe]- hydrogenase is the main enzyme activated in cyanobacterial strains under anoxic conditions [18].
Hydrogenase reabsorbing H2 produced by N2ase is one of the major constraints to the efficient conversion of solar en- ergy into H2 [2,121]. Hup-H2ase catalyzes the almost unidi- rectional uptake of H2, whereas Hox-H2ase catalyzes both H2 uptake and production. Generally, heterocystic cyanobacteria have Hup and Hox, though some only have Hup [122]. To improve N2-based H2 production, Anabaena sp. PCC 7120 was selected as a model strain [123] as its entire genome sequence is available. Since Anabaena sp. contains two types of H2ases, targeted gene disruption was used to inactivate each type and both H2ases, resulting in single (DHup and DHox) and double (DHupDHox) mutants [124]. In an argon environment, inacti- vating Hup activity increased the H2 production rate by 4e7- fold relative to wild-type cells, whereas inactivating Hox ac- tivity did not affect H2 production. Hup-damaged mutants have also been shown to increase H2 production in another Anabaena and Nostoc sp [125].
Since O2-resistant [NiFe]-H2ases in R. eutropha are always inactive, studies suggest that the NiB genes are responsible for tolerance. Instead, structural changes in proximal [FeS] clus- ters facilitate O2 transport. The crystal structure of [NiFe]- H2ase bound to an O2-resistant membrane from R. eutropha H16 has a high resolution [119]. A new [3Fe4S] cluster coordinated to six S atoms of cysteine residues, two of which are identical to O2-resistant hydrogenases, is responsible for O2 tolerance [126]. When Lenz et al. [127] substituted glycine for conven- tional cysteines, the enzyme became more sensitive to O2. Asa result, the previously unique proximal [3Fe4S] cluster was converted to the normal [4Fe4S] cluster. In addition, all future research should concentrate on the proximal [FeS] cluster and its probable significance in determining O2 sensitivity.
A chemical mutagenesis study was conducted using
mutant strains of the filamentous N2-fixing cyanobacteria Nostoc punctiforme PCC 73102, Anabaena variabilis ATCC 29413, and Nostoc PCC 7422 to increase H2 production by several folds [69,76]. Some authors [22,128] claim that changes in H2ase activity or functional relationships with the N2ase complex in the mutant cells are linked to a general regulatory system that regulates cell differentiation. Furthermore, H2ase is influ- enced by a disruption of ETC linked to active N2 and H2 metabolism. Masukawa et al. [129] compiled the amino acids dc-Q193S and dc-R284H, which are in proximity to the [FeMo] cofactor of N2ase, and described mutants of Anabaena sp. PCC 7120 that lacks Hup-H2ase. The wild DHup strain had a lower

 

optimal temperature for H2 production, and unlike the parent strain, H2 splitting in the mutant strain was not completely inhibited in the N2 gas phase. From a practical standpoint, this is very promising, as it authorizes utilizing a less expensive gas phase instead of argon in the process. A defective Syn- echocystis sp. PCC 6803 M55 mutant in the ndhB gene with limited Hup-H2ase activity also produced a higher H2 yield [73]. In the case of N2 fixation, H2ase is involved in processing the H2 produced by N2ase. Therefore, H2ase elimination can potentially yield a higher H2 volume when considering and analyzing cyanobacterial metabolism. For a long time, genetic research on this alteration has been conducted and described in numerous papers. In particular, the gene knockout of the regulator HupR protein reduces HupSL-H2ase expression and
thus increases H2 yield [130].
A possible technique for improving photobiological H2 production under oxygenated conditions is starting with parent strains with high H2ase activity and further inactivat- ing Hup activity. The acetylene reduction assay revealed that Nostoc sp. PCC 7422 had the highest H2ase activity among the 13 heterocystic strains tested [94]. Following the identification of the nucleotide sequences of Nostoc sp. PCC Hup-encoding genes, a Hup-minus (DHup) mutant was engineered by inser- tion and hupL disruption. When grown in the original head-
space over Ar þ 5% CO2 gas under constant light, DHup mutant
cultures collected H2 at rates of 20e30% (w/w) with O2 evolu- tion. H2 accumulation was reduced by <20% when 20% O2 was added to the initial airspace of the DHup cultures, indicating that the mutant H2ase is not sensitive to O2. At incoming light energy of 70 mmol photons m-2 s-1, the DHup mutant con-
verted light energy to H2 with a high efficiency of 1.8% relative
to total solar radiation. Under lab (optimal) conditions, more than 1% of efficiencies relative to total solar irradiance have been reported [129]. However, this was achieved only at a low light intensity, while with increasing light intensity, efficiency decreases significantly, as demonstrated for the DHup mutant PCC 7120 [129,131,132]. The maximum efficiency under bright sunlight was around 0.1% [133]. Targeted mutagenesis to lower the quantity of light-harvesting complexes and/or the concentration of reaction centers, as well as a selection of wild-type strains with greater tolerance to high light intensity, might be used to solve the problem of low light saturation in cyanobacteria.

Engineering O2-resistant mutants
Manipulating O2-resistant H2ase genes is another approach for cyanobacteria-based H2 production [134]. Many studies [135e137] aim to increase H2 production by replacing or de- leting hydrogenase structural genes. The primary goal of recent H2ase studies has been to solve problems associated with increasing H2 production by identifying, copying, and cutting genes not sensitive to O2 to maintain stable H2 pro- duction in cyanobacteria [138]. Many studies have been con- ducted, even though the results are minimal [139,140]. In addition, several recombinant cyanobacterial species with O2 resistance have been obtained. Thus, some of the structural and auxiliary plasmid genes required for the synthesis of H2ase in Thiocaspa roseoperscina were introduced into Syn- echococcus PCC 7942 [141]. One of the most important funda- mental studies is incorporating [Fe]-encoding structural genes
of H2ase into S. elongatus PCC 7942 and synthesizing a func- tional H2-producing enzyme. In addition, two recombinant structural H2ase genes from Synechocystis PCC 6803 were ob- tained using Rubrivivax gelatinosus [142]. When such mutant cyanobacterial strains with high production of O2-resistant H2ase are developed and employed, it is unnecessary to aerate the culture medium with an inert gas to induce anaerobic conditions.
The main properties of oxygen-resistant enzymes are directly related to the metals they contain. The unique cluster [3Fe4S] not only ensures O2 transport through the H2ase bound to the R. eutropha membrane but also suggests that auxiliary HoxR and HoxT proteins may be involved in O2 transport [119]. The disruption of HoxR or HoxT genes leads to an increase in O2 sensitivity and a change in the unique [3Fe4S] cluster composition, as demonstrated experimentally. The predictive corresponding HoxR functions in forming a unique [3Fe4S] cluster are inconsequential for cofactor sta- bility development.
Obtaining O2-insensitive mutants in cyanobacteria is currently an essential issue, so research in this direction will undoubtedly be of interest. In this context, HoxR and HoxT proteins from R. eutropha are important factors that need further investigation.

Hydrogenase behavior in an anoxic environment
Accidental gene insertion (hox, hyp, hup) can increase the ac- tivity of the natural enzyme H2ase. Neighboring relatives of cyanobacteria or other prokaryotic microorganisms act as donors. Research has been conducted on plasmid introduc- tion into the cytoplasm by cross-cutting cells. Related studies [143,144] reported that the rate of H2 production in the [FeFe]- H2ase encoding strains C. acetobutylicum and C. saccha- robutylicum increased by 67% on average compared to the natural strain. In addition, the Synechococcus elongatus strain UTEX 2973 showed a relatively higher H2 yield. It co-existed with C. saccharobutylicum in nature, and the sequencing showed that this cyanobacteria strain contains a unique [FeFe]-H2ase gene responsible for H2 production. H2ase activ- ity was found to be 1.3-fold higher in this study. The utility of such strategies in producing O2-sensitive or non-O2-sensitive mutant species is enormous, and all genetic studies aimed at increasing H2 production use cyanobacterial [NiFe]-H2ase and N2ase enzymes.
Active species containing new and unique hydrogenases
with the functions and properties required for H2 production can be isolated from the environment, particularly in cells living in specific environmental conditions (extreme regions). For example, numerous changes in [FeFe]-hydrogenase adapted to living in an O2-free environment have been observed in studies of the H2ase enzyme diversity. It was discovered that strictly conservative amino acids undergo distinct changes in redox properties and active site O2 sensi- tivity. Similar methods can be used to create effective and non-O2-sensitive H2ases, such as [NiFe]-H2ase [145]. Ideas for genetic engineering research can be obtained by studying microorganisms that have altered their properties to survive and adapt to such natural conditions.
Moreover, research in this realm involves expressing and
introducing O2-resistant [NiFe]-H2ases into the cell. For

 

example, HydSL H2ase expressed by the sulfur bacterium purple Thiocapsa roseopersicina was incorporated into unicel- lular Synechococcus sp. PCC 7942 [141]. However, the introduced foreign [NiFe]-H2ase requires an extra maturation system and a consistent electron supply for H2 production. Therefore, the O2- and heat-resistant NiFe-HynSL H2ase (HynSL) was incor- porated into the S. elongatus chromosome along with 11 indi- rect Atheromonas macleodii genes [141]. As a result, a 3-fold higher H2 production was obtained in the recombinant strain. Genetic engineering accomplishments also include enhanced expression of the inserted genes, O2 tolerance, and effective integration of the H2ase into the photosynthetic
ETC [16].

Incorporation of heterologous hydrogenase into cyanobacteria cells
The N2ase or natural bidirectional [NiFe]-H2ase activity is tightly linked to cyanobacteria-based bio-H2 production. However, H2 production with natural enzymes remains very low and far from economic productivity. Therefore, genetic engineering studies need to be conducted, albeit at high in- tensity, for which the host cell and primary enzymes are necessary. All N2ases have a low cycle number and require 2 ATP molecules to reduce one electron. In addition, they secrete at least 1 mol of H2 for each mole of N2 absorbed [146]. In cyanobacterial cells, uptake, or bidirectional [NiFe]-H2ase, degrades H2 molecules in relatively small amounts [147]. In contrast, H2 production based on [NiFe]-H2ase is not depen- dent on the ATP molecule and has a high rate. [FeFe]-H2ase is a relatively well-studied enzyme for genetic engineering. Therefore, [FeFe]-H2ase can be used as a model enzyme when implementing recombinant expressions in synthetic systems [148]. However, no naturally occurring [FeFe]-H2ase has been discovered in cyanobacteria [66]. Thus, one of the most important strategies is to isolate [FeFe]-H2ase and introduce it into cyanobacterial cells. This enzyme is most commonly found in enzymatically aerobic Clostridium [149]. Another model system is [FeFe]-H2ase (HydA1) from the microalga Chlamydomonas reinhardtii, which consists only of the F- domain and lacks the FeS cluster [150]. The ability of
metabolic [FeFe]-H2ases isolated from microalgae to connect with water oxidation by PSII and photosynthetic ETCs sets them apart. However, the expression of [FeFe]-H2ase in nat- ural cyanobacteria is fraught with complications. This is because cyanobacteria can only produce gene signals that target the maturation of their [NiFe]-H2ase under natural conditions. With the extraction of [FeFe]-H2ase from Clos- tridium sp. and its successful introduction into the unicellular cyanobacterium Synechococcus sp. [151], there has been a small breakthrough in this direction in the last decade. In vitro and in vivo, the heterologous, foreign [FeFe]-H2ase was also active. In vivo [FeFe]-H2ase activity was light-dependent and associ-
ated with ETC reactions. This study showed that AMC414 lacks Hup activity, and increased H2 (3.75 nmol H2 Chl-1 a h-1) was observed compared to the wild type (1.5 nmol H2 Chl a h-1). Again, [FeFe]-H2ase was degraded in filamentous heter- ocystic Nostoc PCC 7120 blue-green algae. The hetR gene, which
is only expressed and functions in heterocystic cyanobacteria, was used as a promoter for this purpose [152]. The [FeFe]- H2ase protein is found not only in cyanobacteria but also in Escherichia coli. The H2 yield increased when [FeFe]-H2ase was delivered into the cell along with HydE, HydF, and HydG genes [153]. However, no biochemical or genetic evidence supports the maturation of [FeFe]-H2ases in cyanobacteria. This research is not limited to bacteria but is also carried out in eukaryotes. It has been shown that the [FeFe]-H2ase of Clos- tridium sp., which lacks the genes responsible for the matu- ration system, is expressed in the cyanobacterium Synechococcus PCC 7942 under in vivo and in vitro conditions [154]. Similar findings were attained in a study on transferring the HydA gene from Chlamydomonas to Synechocystis PCC 6803. Activation of an enzyme transferred into a foreign cell may occur due to the taxonomy of genes copied along with the maturation system. As shown in the preceding study [155], when [FeFe]-H2ase was transferred to Synechocystis sp. PCC 6803, without additional maturation genes, was activated differently in each organism. Why [FeFe]-H2ase is activated in some organisms without maturation operons and why some organisms do not need to express it is an area of research that still requires clarification (Table 2).

 

Table 2 e Hydrogen production in engineered non-native enzymes of cyanobacterial strains using different strategies.
Cyanobacterial strains
Engineered genes
H2 production rate
H2 production assay condition
References
Synechosystis sp. PCC 6803
hydA from Chlamydomonas
130 nmol H2 mg Chl a-1 h-1
Anaerobic; 50 mEm-2 s-1;
[155]

reinhardtii

5 mm DCMU

Anabaena sp. PCC 7120
H2ase operon (hydA, hydB,
3.4 nmol H2 mg Chl a-1 h-1
Light and nitrate
[152]
(Hup-)
hydE, hydF, hydG along with

deprivation

 

two additional genes,

 

 

 

S03922 and S03924, from

 

 

 

Shewanella oneidensis MR-1)

 

 

Synechococcus elongatus
hydA and maturation
2.8 mmol H2 mg Chl a -1 h-1
Light; 5 mm DCMU; bubbling
[151]

operon (hydEFG) from

with 2.5% CO2 and 97.5% N

 

Clostridium acetobutylicum

 

 

Synechococcus elongatus
[NiFe]-H2ase from Thiocapsa
roseopersicina
~0.07 nmol H2 mg protein h-1
Anaerobic; 40 mEm-2 s-1
[141]
Synechococcus elongatus
[NiFe]-H2ase (hynSL along
~4.2 nmol H2 mg protein h-1
Anaerobic; 40 mEm-2 s-1
[141]

with 11 adjacent proteins)

 

 

 

from Alteromonas macleodii

 

 

Synechococcus PCC7942
hydA from C. acetobutylicum
~6.8 mmol H2 mg Chl a-1 h-1
Ar, dark adaptation
[154]

 

Studies on [FeS] cluster ligation in [NiFe]-H2ases small subunits have recently changed the enzyme proclivity to accept H2ases. Yonemoto et al. [156] developed an O2-tolerant enhanced H2 production scheme in cyanobacterial strains by introducing site-directed mutations into A. macleodii O2- tolerant [NiFe] HynSL hydrogenase, a membrane-associated H2ase. Due to its 2% O2 tolerance, this H2ase is appealing for biotechnological applications. The ligand modification was based on the mutagenesis of uptake H2ase from D. fructoso- vorans [157]. Yonemotto et al. [158] also continued the inves- tigation of A. macleodii H2ase to obtain an enzyme with considerably higher H2 production activity than previously reported. The substitutions in A. macleodii HynSL were thor- oughly investigated, with aspartic acid, histidine, asparagine, or glutamine substituted for each of the 12 amino acid sites linking three [FeS] clusters in the small subunit, as well as alternate coordinating ligands.
In general, all H2ase use direct or auxiliary mechanisms to
transport electrons to the donor, i.e., Fdx. An unanswered question is whether the functional foreign [FeFe]-H2ase can effectively interfere with the main metabolism, specifically whether it can transfer or accept electrons to the cell redox zone. Artificial [FeFe]-H2ase introduction into the whole redox system and complete contact with the host cell is a very questionable process. This statement is supported by research on Synechococcus elongatus [151]. The electron transfer to the introduced [FeFe]-H2ase and the energy stored in reserve are handled differently by distinct Fdx. As a result, Fdx that can modulate the introduced foreign H2ase into the cell must be created. Consequently, continuous electrons can reach the introduced artificial [FeFe]-H2ase.
Cyanobacteria-based H2 production can achieve higher
yields with O2- and heat-resistant enzymes. This might offer benefits over O2-labile [FeFe]-H2ases.

Engineering a photosynthetic apparatus to enhance H2 production

The mechanism of photosynthesis evolved over millions of years to operate under unfavorable environmental conditions and competition, but not as efficiently as possible. The ratio of PSI and PSII, linear-cyclic electron transport, the volume of the LHC, and the number of cells all play essential roles in the efficient operation of the photosynthetic process [110]. The traits mentioned above can be genetically engineered to improve the photosynthetic structure operation. Cyanobac- teria have a lower PSII/PSI ratio than microalgae and plants, resulting in better cyclic electron transport performance than linear electron transport [159]. Many mutant strains were ob- tained to increase the H2 yield by enlarging the antennae. For example, the mutant Olive (apcE and apcAB operons deleted) cyanobacterium Synechocystis sp. PCC 6803 without phycocy- anin antennae produced more H2 than the wild species [160]. As a result, the cyanobacterial species do not consume redundant energy sources, leading to an increment in PSI/PSII ratio. Ac- cording to the findings, the Olive mutant is an encouraging H2 production contender. An increase in the pool of photosyn- thetically derived NADPH regenerators and linear electron transport between photosystems of the Olive mutant were
weakened by the pigment phycocyanin [160]. Moreover, to compensate for the excessive reduction in cellular metabolism, the mutant strain enhanced the amounts of oxidative and os- motic stress-related enzymes and carbon fixation. It can be concluded that the mutant Olive, whose phycocyanin pigment was removed, is the most promising candidate for further ge- netic modification as a constructive cyanobacterial cell [161].
Indeed, genetic engineering methods are being used to reduce phycobilin pigments and chlorophyll antennae to in- crease cyanobacteria-based H2 yield. As only two photons are required for the transfer of electrons acquired from the water decomposition, theoretically, at high light intensities, a cell PSA absorbs and wastes fewer photons. Therefore, engineer- ing mutants by reducing the quantity of light-absorbing pig- ments in the PSA allows for a 10% efficiency in turning solar energy into carbohydrates. Moreover, H2 can be obtained from these cells [162]. Bernat et al. [160] demonstrated that continuous growth of a mutant Synechocystis PCC 6803 with
shortened or reduced antennae could produce 200 mL H2 h-1
culture. Linear electron transport was 6-fold faster in a
Synechocystis PCC 6803 mutant missing a phycobilisome than in a wild strain, whereas cyclic electron transport was the same in both strains. The main prerequisite for increasing H2 production under conditions of increasing sunlight absorption is an enhancement of the linear electron flow rate.
Recently, it was discovered that protein synthesis tech- nology could significantly improve photosynthetic electron transport efficiency to the H2ase. To prevent competing with Fdx-NADPþ oxidoreductase (FNR), Fdx can be combined with
H2ase [163]. Eilenberg et al. [117] found that combining [FeFe]-
H2ase with Fdx in vivo increased photosynthesis 4-5-fold and improved O2 resistance over wild-type [FeFe]-H2ase. Alterna- tively, PSI can also directly combine [FeFe]-H2ase. Kanygin et al. [52] used this approach to develop the PSI H2ase chimaera, which is integrated into the HydA chain unit. Under
anoxic conditions, the engineered C. reinhardtii strain pro- duced stable H2 for 5 days at a rate of 14 mmol H2 mg Chl-1 h-1. Moreover, H2 photoproduction occurs independently of the
Calvin cycle in nutrient-rich algae.
Cyanobacteria Acaryochloris marina having a high Chl d con- tent could be another impressive H2 producer. Its plasmid (pREB4) contains the hypABCDEF gene, which encodes a com- plete set of bidirectional H2ase subunits [164]. A. marina can utilize light until the near-infrared region, as the absorption maximum of Chl d is near red. A. marina is a natural mutant because its unique redox potential of electron-carrier compo- nents in photosynthesis is directly tied to it. One of the fundamental studies is the study of H2 splitting under the red color spectrum of a strain with unique photosynthetic pig- ments [165].
Berna't et al. [160] used Synechocystis PCC 6803 strains with
genetically modified antennae to produce H2. The authors made a potential hydrogen production strain using antenna- deleted Synechocystis sp. PCC 6803, and their results showed that decreasing or missing PBS antenna gave the mutant strains (Olive and PAL mutants) the capability to grow under limited light. Also, the group showed that the linear electron transport rate increased by 1.5-fold in PC- and PBS-depleted Olive and PAL mutants. Furthermore, the effects of the strain on the ETC and the environment were determined.

 

When photoautotrophic mutants with shortened phycobili- some antennas were grown under the light, they produced the most H2 (200 mL H2 h-1 by a 1 L culture).
Numerous literature data cover H2 production pathways in
anoxygenic organisms and their relevance to photosynthesis [166e169]. However, further studies are needed to increase the efficiency, productivity, and reliability of photosynthetic H2 production in direct solar energy conversion.

Effects of resistance to oxidative and sugar stress

Cyanobacteria are of global interest as biological instruments for carbon capture and storage. They are considered pro- ducers of sustainable biofuels due to their excellent photo- synthetic endowment. Their genetic simplicity and physical stability may one day make them the most important indus- trial species. A well-developed complex group of enzymes is expected to contribute to the current energy deficit, making cyanobacteria-based H2 production a promising industry [170].
With each study, the structure of the cyanobacterial enzyme types involved in H2 production becomes more complex [39]. Therefore, the strain Synechocystis PCC6803, whose genome has been fully sequenced, was used. A bidi- rectional pentameric H2ase that is reversibly O2-inactivated
can produce H2. For photosynthesis and/or sugar catabolism, the H2 reaction (2Hþ þ 2e- 4 H2) relies on electrons from NAD(P)H, as well as redox cofactors from the [NiFe] center and
multiple [FeS] clusters. The HoxEFUYH operon is controlled by a feeble promoter [171] expressed by 168 bp in the upper stream of the proximal hoxE gene codon. Meanwhile, the HoxEFU subunits work together to form a diaphorase compo- nent, which transports electrons to the HoxHY H2ase unit, which uses NAD(P)H to produce H2 from protons. Physiolog- ical studies have revealed that H2ase operates as an emer- gency electrical valve, releasing extra photosynthetic electrons, such as switching from (anaerobic) dark to light conditions, resulting in debilitated and temporary H2 production.
According to Ortega-Ramos et al. [171], overexpression of the HoxEFUYH operon protects cells from stress caused by H2O2, glycerine, and glucose. Simultaneous overexpression of the HoxEFUYH and HypABCDEF proteins resulted in a 20-fold increase in H2ase activity. The authors concluded that the increased cellular production of H2 proteins positively affects H2 yield and that future increases in H2 yield can only be achieved through genetic engineering.
Germer et al. [103] used the psbAII promoter to increase H2ase activity in Synechocystis sp. via the expression of the endogenous hoxEFUYH operon of strain Nostoc PCC7120 and the heterologous NoshypABCDEF operon. As a result, the
mutant strain H2 yield increased from 2.9 (wild type) to 9.4 (mutant) nmol H2 mg Chl a-1 min-1 [103].
Despite the preceding investigations, H2 metabolism re-
mains a mystery. More profound knowledge is required to identify favorable environmental conditions and to carry out powerful genetic alterations to speed up the rate of H2 pro- duction [172]. Obtaining mutants of cyanobacteria that are resistant to sugar and various salts, in turn, opens the way to
rapid biomass production and increases the H2 yield. This is because the amount of biomass is vital in obtaining any bio- logical component.

Developing a zero-waste H2 production technology using genetically engineered cyanobacteria

A promising future research direction is the development of a zero-waste H2 technology based on cyanobacterial strains. Many technologies are currently proposed to produce bio- diesel and bioethanol from cyanobacteria [170], but there are no plans yet to develop a zero-waste energy technology based on cyanobacterial H2. According to the current envi- ronmental policies, one of the most important activities is to develop energy production technologies that do not pollute nature.
Nutrients (macro- and microelements) are extracted from municipal wastewater (1) to cultivate mutant strains, as shown in Fig. 3. This technology is implemented by con- structing special photobioreactors in manufacturing areas. Household or industrial wastewater is treated in two stages:
(2) before being purified in natural ponds and (3) until entering the photobioreactor. The conditions for H2 production are then created, and the nutrients are transported into the pho- tobioreactor, where the cyanobacteria grow. Since the pho- tobioreactor is located in an external environment (4), solar energy is sufficient. The CO2 required for cell growth is transported via special hoses from nearby manufacturing fa- cilities and fed directly or indirectly to the photobioreactors
(5). The H2 energy produced is stored in special H2 cylinders
(6). The resulting pure H2 energy is stored in bacteria (7) as electrical energy or used for lighting and motor vehicles (8). Hydrogen energy can also be used directly as a high-power energy source in the heating system (9) and the space in- dustry (10).
As shown in Fig. 3, in the future, by creating smart cities, it will be possible to reduce the current main problem - the temperature of the Earth's surface. At the same time, the technologies of waste remediation, reuse of wastewater, and creation of new, inexhaustible energy sources can be consid- ered the main plan to prevent the energy crisis. Furthermore, by constructing the photobioreactors for growing cyanobac- teria in production facilities, industrial CO2 can be directly used for cell growth. In this context, one of the future genetic research's main goals is to create new strains capable of actively growing and releasing H2 energy in an environment with high CO2 (1e10% of air).
The main advantage of the cyanobacteria-based H2 pro- duction technology is higher biomass productivity. However, since cell division occupies an important place in the pro- duction of any biological product, it is also critical that the mutant strain's growth intensity is high for H2 energy. Table 3 summarises the most important characteristics of the cya- nobacterial strains for H2 production technology.
However, culturing cyanobacterial strains in wastewater has its challenges. This is because wastewater contains very high amounts of lignocellulosic materials (LMs) [173]. LM is a composite based on cross-linked biopolymers on a dry basis,

 

Fig. 3 e Zero-waste technology for H2 production based on genetically modified cyanobacteria. Note: 1 e municipal wastewater; 2 e treatment facilities; 3 e aero tank; 4 e open bioreactor for cyanobacterial biomass; 5 e transfer of CO2 from flue gas of thermal power plants to the bioreactor; 6 e H2 storage; 7 e storage of H2 generated electricity; 8 e the electricity generated can be used in the transport industry and as a light source; 9 e H2 can be used as an energy source for heating generators; 10 e H2 can be used as the main energy source in the space industry.

 

with 35e45% cellulose, 25e30% hemicellulose, and 25e30% lignin [174]. These are classified into four main proportions based on their source, namely woody biomass, agricultural waste, energy crops, and cellulosic waste groups [175]. It is likely that LMs negatively affect the growth of cyanobacterial cells and lead to a decrease in H2 content. However, using LMs as biofilms, their toxicity can be eliminated, and cell growth is stimulated. For example, Zhang et al. [173] made the first attempt to develop a new biofilm technology using LMs as biofilm carriers. The authors selected LMs pine sawdust, rice husks, sugar cakes, and oak sawdust, which are produced in large quantities around the world every year. Finally, the toxicity of LMs to cells was determined by measuring the growth parameters and chemical composition of C. vulgaris grown in biocarrier leachate. Next, Zhang et al. [176] obtained five different LMs and used microalgae strains for their utili- zation. The effect of the physicochemical properties and toxicity of biocarriers on the growth and attachment of cells was determined. C. vulgaris cell growth was promoted by pine sawdust filtrate (15.5%), whereas shoot bark filtrate (15.5%) and sugarcane (13.2%) inhibited cell growth during light cultivation.
Phototrophic cells attach to LMs and use them as natural materials for biofilm formation. After cultivation, the cyano- bacterial biomass can be harvested together with LMs or separately for disposal [177]. Interestingly, the collected mixture (LMs and cyanobacterial biomass) can be used directly in the laboratory or industrial conditions as a raw material for H2 production. However, although the use of LMs as carriers in industrial photobioreactors is economically beneficial, the following conditions should be taken into ac- count first.
Choosing the optimal materials for fixing the cells that increase the efficiency of the new technology;
Assessment of the chemical composition of the collected cyanobacterial biomass;
Determining the effect of material surface properties on algae productivity;
Identification of factors that positively affect the growth of cyanobacterial cells.

The abundance of fossil fuels, which is still running out, is the primary reason for the lack of development of biological H2 energy. Since the cost of implementing investment projects is so high, many countries purchase cheap fossil fuels to compensate for household needs. Despite intensive study, bio-H2 production is lagging, and most H2 produced is derived via cracking.

Challenges and future perspectives

Although the mechanics behind the primary steps of H2 pro- duction by phototrophic microbial cells are fully established, bio-H2 production is still a long way from being implemented. The main constraints for sustainable H2 production are the enzyme O2 sensitivity and electron competition across distinct pathways that use reduced electrons. Therefore, cyanobacteria-based H2 production has only been observed in lab conditions thus far. To reach an industrial scale, the pace and duration of H2 production must be increased. Many alternative ways are being investigated to increase H2 pro- duction. These techniques have three major components, namely, metabolic, genetic, and technological (Fig. 4) [33,34].

Uses only regenerating substrates: sun and
water
High growth rate
Flexible to genetic modifications Can grow on cheap salts
Independent of geographical location and weather conditions
Easy to handle
Expensive photobioreactor materials Reliant on electricity
Difficulty in separating H2 and O2 The complexity of H2 gas storage technology
High transport costs Lack of supporting infrastructure in the fuel industry
Large-scale development of mutants with higher H2 yields
The necessity of high-level governmental and international programs
Launch of high-level H2 capture and storage projects
Granting of rebates for the transport of bio- H2 energy
High cost of bio-H2 energy Lack of international support
Low demand for non-renewable energy sources
Inability to withstand competition
Strengths

 

 

 

Weaknesses

 

 

 

Opportunities

 

 

 

Threats
Table 3 e SWOT analysis of zero-waste H2 production technology [2,19].
The emerging demand for the development of sustainable solutions to utilize solar energy with the subsequent produc- tion of energy sources for the social community arouses sci- entific interest [15]. However, along with solar cells to produce electricity, there are other ways to produce solar fuel. One of the most challenging fields of biotechnology to tackle envi- ronmental concerns is the development of photosynthetic cyanobacterial cells to generate sustainable solar fuels. Native and photosynthetic cyanobacteria have long been utilized as model systems to investigate, demonstrate, and develop photobiological H2 production. However, the level of H2 pro- duction attained is negligible from the perspective of indus- trial production of competitive energy carriers. Therefore, solutions to boost bio-H2 production in cyanobacteria must be discovered through research. As shown in Fig. 4, recent breakthroughs in genetic engineering, which employs syn- thetic biology to engineer a cyanobacterial cell to produce the desired product, such as H2, open up new possibilities andalternatives.
High-throughput assays and screening technologies, along with computational and systems biology breakthroughs, have enabled the examination of vast volumes of data to predict cell metabolism and physiology at the systems level. In addition, synthetic biology will enable the production of spe- cifically tailored cyanobacterial cells capable of producing considerable volumes of renewable H2 [97].
To investigate an organism at the systems level, systems biology can utilize a number of genome-wide technologies,

Fig. 4 e Prospects for cyanobacteria-based H2 production using metabolic engineering systems to design and create cyanobacterial strains that convert solar energy and water directly into the preferred solar fuel, H2.

 

such as high-throughput analytical methods and computa- tional and ohmic studies encompassing the genome, tran- scriptome, proteome, and metabolome levels. The findings of such studies can be used extensively in strain construction using metabolic engineering. Generally, the entire procedure is repeated until the desired phenotype and performance are achieved. Based on in silico modeling, knockouts and inserts will be possible in the future. The development of synthetic biology will allow the creation of modified cyanobacterial cells with predicted responses and/or the development of bio- reactors. Still, it will also have implications for economic costs, public acceptance, and the development and use of H2 energy systems in our society. Due to their rapid growth and simplicity, Cyanobacteria offer an ideal foundation for ambi- tious metabolic engineering projects.

Conclusion

Hydrogen production has only been determined on a labora- tory scale thus far, as the yield remains insufficient for the industrial scale. Consequently, to regulate the H2 production rate, the design and operational parameters must be opti- mized for maximal H2 yield. Currently, efforts to increase the H2 production capabilities of cyanobacteria species are carried out mainly in four directions: modification of nitrogenase enzyme genes, obtaining hydrogenase mutants, increasing electron transport by changing the photosynthesis complex and obtaining mutants resistant to external environmental factors. However, due to the low H2 yields of the obtained mutants and the high costs involved, their introduction into production is ineffective. In this context, developing a waste- free technology using a genetically modified active H2 pro- ducer is of great importance. The zero-waste technological system realizes hydrogen production and contributes to the reduction of the price of bio-H2 in the future. Furthermore, the construction of zero-waste technology with mutant species in the production of bio-H2 will not only alleviate future energy concerns but also lower CO2 levels in the environment.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research has been funded by the Ministry of Science and Higher Education of the Republic of Kazakhstan under these projects: AP13067574, AP14869048, and AP09260077. SA
gratefully acknowledges support from the Distinguished Sci- entists Fellowship Programme, King Saud University, Saudi Arabia. JCWL, JSC and SIA were supported by the joint grant RSF-MOST (22e44-08001). JSC gratefully acknowledges the financial support from the Taiwan Ministry of Science and Technology (grants MOST-110-2221-E-029-004-MY3, 111-3116-
F-006 -004, 111-2621-M-029 -002). The present work was also financially supported partly by Grants-in-Aid for Scientific Research (No. 21K06101 (T.T.)). Fig. 3 was supported under the state contract of the Ministry of Science and Higher Education of the Russian Federation (Project No. 122050400128e1).

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