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1. (WO2019033095) IMPROVED IN VITRO TRANSCRIPTION/TRANSLATION (TXTL) SYSTEM AND USE THEREOF
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CLAIMS

1. A composition for in vitro gene expression, comprising:

a treated cell lysate derived from one or more host cells such as bacteria, archaea, plant or animal;

a plurality of supplements for gene transcription and translation;

an energy recycling system for providing and recycling adenosine triphosphate (ATP); and

one or more exogenous additives selected from the group consisting of polar aprotic solvents, quaternary ammonium salts, sulfones, ectoines, glycols, amides, amines, sugar polymers, sugar alcohols, slow elongation-rate RNA polymerase (RNAP) and ribosomes, wherein preferably the sugar polymers and sugar alcohols are not for providing energy source.

2. The composition of claim 1, for use in expressing a metagenomically derived gene, a plurality of genes that together constitute a pathway, and/or synthetic proteins, wherein preferably the pathway is designed for synthesis of a natural product.

3. The composition of claim 2, wherein the gene or pathway has not been optimized for in vitro gene expression.

4. The composition of claim 1, wherein the plurality of supplements comprise magnesium and potassium salts, ribonucleotides, amino acids, a starting energy substrate, and a pH buffer.

5. The composition of claim 1, wherein the one or more additives modulate nucleic acid secondary structure, improve RNAP processivity and/or stability, affect RNAP elongation rate, improve ribosome synergy with RNAP and/or stability, and/or improve stability of polypeptide being synthesized.

6. The composition of claim 1, wherein the slow elongation-rate RNAP is homologous to the host cells, such as RNA Poll, RNA PolII, RNA PolIII, and bacterial RNAP.

7. The composition of claim 1, wherein the slow elongation-rate RNAP is heterologous to the host cells, such as SP6 RNAP variants, T7 RNAP variants, and T3 RNAP variants.

8. The composition of claim 7, wherein the slow elongation-rate RNAP is sourced from a

thermophile or psychrophile.

9. The composition of claim 1, wherein the slow elongation-rate RNAP is a synthetic RNAP such as engineered T7 RNAP variants and engineered RNA PolII variants.

10. The composition of claim 9, wherein the slow elongation-rate RNAP is engineered by directed evolution and/or rational design.

11. The composition of claim 1, wherein the slow elongation-rate RNAP is provided as a purified protein or as a nucleic acid encoding the slow elongation-rate RNAP.

12. The composition of claim 1, further comprising exogenous nucleic acids to be expressed in the composition, wherein each exogenous nucleic acid comprises a promoter that is recognized by the slow elongation-rate RNAP.

13. The composition of claim 1, wherein the ribosomes are sourced from the host cells, or from an organism different than the host cells, wherein preferably the ribosomes are provided at 0.1 μΜ to 100 μΜ concentration.

14. The composition of any one of claims 1-13, wherein the composition comprises both slow elongation-rate RNAP and exogenous ribosomes, wherein preferably the slow elongation-rate RNAP and the exogenous ribosomes are coupled, wherein optionally such coupling is orthogonal to the host cells.

15. A method of preparing the composition of any one of claims 1-14, comprising:

providing an in vitro transcription/translation system comprising the treated cell lysate, the plurality of supplements and the energy recycling system; and

supplying the one or more exogenous additives.

16. A method of in vitro gene expression, comprising:

providing the composition of any one of claims 1-14, and

providing one or more nucleic acids to be expressed.