Low-Carbon-Footprint Production of High-End 5-Aminolevulinic Acid via Integrative Strain Engineering and RuBisCo-Equipped Escherichia coli

Abstract

5-Aminolevulinic acid (ALA) is an imperative compound that has been widely applied in the agricultural and medical fields. The ALA synthesis in Escherichia coli via the C4 pathway depends on ALA synthase (ALAS) and releases CO2 in a single-step reaction. However, enhancement of the ALA production by increasing the protein’s quality and quantity with simultaneous CO2 reduction has never been reported. In this study, the expression and activity of ALAS were improved by employing chaperone and rare tRNAs. Moreover, the pdxY gene was introduced for pyridoxal phosphate (PLP) regeneration, a cofactor for ALAS, and significantly increased ALA production to 14.3 g/L through stepwise strain engineering with an optimal feeding strategy. The leading excess CO2 emission was further reused as a second carbon source for cell growth and ALA synthesis by cloning the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) and phosphoribulokinase (Prk) in E. coli. As a result, the co-application of RuBisCo, Prk, and ALAS reduced CO2 emission by 53.8% in combination with pdxY and pRARE. Finally, the crude ALA was demonstrated and applied in the antibacterial photodynamic therapy (aPDT) against three different pathogens. The robust strain, RSSA-BY with a lower carbon footprint, achieved the highest cell growth and efficient ALA production for aPDT for the first time, providing a vital and green bioprocess toward an advanced and sustainable future.

Reprogramming T7RNA Polymerase in Escherichia coli Nissle 1917 under Specific Lac Operon for Efficient p-Coumaric Acid Production

“…Recent studies coupled GroELS in the T7 system for remarkable improvements in solubility, catalytic activity, and thermostability of recombinant enzymes. ,,, Except for RtTAL expression from plasmid or chromosome in ET7L strain (i.e., RRtL and L::Rt), three additional constructs were designed with the GroELS gene, namely, RRtGL (RtTAL and GroELS in one plasmid), RRtL::G (integrated GroELS to ET7L genome at the HK022 site and harboring RRt plasmid), and L::RtG (integrated RRtG to ET7L genome at the HK022 site) (Figure A). The protein expression of genetic strains was tested under LB medium and is presented in Figure B.…”

“…The recombinant plasmids were then transformed into ET7L (L) and ET7H (H) strains as PRtL, RRtL, PRtH, and RRtH. For GroELS under plasmid design, GroELS was inserted into the downstream of RtTAL in pSIT vector and further transformed into ET7L strain, named RRtGL plasmid . On the other hand, RRt plasmid was also overexpressed using L::G strain and known as RRtL::G.…”

“…The flow rate was maintained at 0.4 mL per minute. 9 Meanwhile, the analysis of pCA titers was equipped with a YMC-Triart C-18 column (250 × 4.6 mm), and an UV detector at 310 nm was used to analyze. A dualsolvent, acetonitrile−water containing 0.1% formic acid, was employed for the separation of proteins using a gradient system at a flow rate of 1 mL/min over 30 min.…”

“…T7RNAP and the cognate T7 promoter have been broadly employed as an efficient system for high-level recombinant expression in E. coli DE3 strain. ,, The high orthogonality of T7RNAP and T7 promoter rendered an energy competition between the T7 system and the housekeeping proteins, thus triggering metabolic burden and hampering cell growth . Hence, fine-tuning of T7RNAP expression was inevitably required to optimize production by varying the genetic elements, including promoter strengthens (e.g.,E.…”

“…It consists of three structural genes: lacZ encodes β-galactosidase for lactose degradation, while lacY and lacA encode lac permease and lac transacetylase, respectively. As the first well-understood genetic regulatory mechanisms in Escherichia coli, the polycistronic transcripts of lac operon became a paradigm for gene regulation in other prokaryotes, such as Bacillus subtilis, Lactococcus lactis, Streptococcus pyogenes, and Lactobacillus delbrueckii. − The lac operon regulates lactose transport and utilization by coordinating transcription under the control of two regulators, lac repressor and catabolite activator protein, that switches the operon between “on” and “off” mode in response to lactose and glucose levels, respectively. , In particular, the lac promoter is used for recombinant protein production and has been adopted for the optogenetic control and production of chemicals and toxic proteins. − Till date, lac operon has been widely utilized in combination with the T7 system, in which a T7RNA polymerase (T7RNAP) is regulated by the lac I/ lac O system to amplify the production of functional proteins controlled under the T7 promoter …”